Interleukin-1 (IL-1)
Interleukin-1 (IL-1.alpha. and IL-1.beta.) is a "multifunctional" cytokine that affects nearly every cell type, and often in concert with other cytokines or small mediator molecules. (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15, 1996).) There are three members of the IL-1 gene family: IL-1.alpha., IL-1.beta., and IL-1 receptor antagonist (IL-1Ra). IL-1.alpha. and IL-1.beta. are agonists and IL-1Ra is a specific receptor antagonist. IL-1.alpha. and .beta. are synthesized as precursors without leader sequences. The molecular weight of each precursor is 31 kD. Processing of IL-1.alpha. or IL-1.beta. to "mature" forms of 17 kD requires specific cellular proteases. In contrast, IL-1Ra evolved with a signal peptide and is readily transported out of the cells and termed secreted IL-1Ra (sIL-1Ra).
IL-1 Receptor and Ligands
The receptors and ligands of the IL-1 pathway have been well defined (for review, see Dinarello, C. A., FASEB J. 8:1314-1325 (1994); Sims, J. E. et al., Interleukin-1 signal transduction: Advances in Cell and Molecular Biology of Membranes and Organelles, Vol. 3, JAI Press, Inc., Greenwich, Conn. (1994), pp. 197-222). Three ligands, IL-1.alpha., IL-1.beta., and IL-1 receptor antagonist (IL-1Ra) bind three forms of IL-1 receptor, an 80-kDa type I IL-1 receptor (IL-1R1) (Sims, J. E. et al., Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor (IL-1RII) (McMahan, C. J. et al., EMBO J. 10:2821-2832 (1991)), and a soluble form of the type II IL-1R (sIL-1RII) (Colotta, F. et al., Science 261:472-475 (1993)).
IL1 Production in Various Disease States
Increased IL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors; leukemias; Alzheimer's disease; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic diseases (myocardial infarction); noninfectious hepatitis; asthma; UV radiation; closed head injury; pancreatitis; periodontitis; graft-versus-host disease; transplant rejection; and in healthy subjects after strenuous exercise. There is an association of increased IL-1.beta. production in patients with Alzheimer's disease and a possible role for IL-1 in the release of the amyloid precursor protein (Vasilakos, J. P., et al., FEBS Lett. 354:289 (1994)). However, in most conditions, IL-1 is not the only cytokine exhibiting increased production and hence the specificity of the IL-1 findings as related to the pathogenesis of any particular disease is lacking. In various disease states, IL- 1.beta., but not IL-1.alpha., is detected in the circulation.
IL-1 in Therapy
Although IL-1 has been found to exhibit many important biological activities, it is also found to be toxic at doses that are close to therapeutic dosages (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15, 1996)). In general, the acute toxicities of either isoform of IL-1 were greater after intravenous compared with subcutaneous injection. Subcutaneous injection was associated with significant local pain, erythema, and swelling (Kitamura, T., & Takaku, F., Exp. Med. 7:170 (1989); Laughlin, M. J., Ann. Hematol. 67:267 (1993)). Patients receiving intravenous IL-1 at doses of 100 ng/kg or greater experienced significant hypotension. In patients receiving IL-1.beta. from 4 to 32 ng/kg subcutaneously, there was only one episode of hypotension at the highest dose level (Laughlin, M. J., Ann. Hematol. 67:267 (1993)).
Contrary to IL-1-associated myelostimulation in patients with normal marrow reserves, patients with aplastic anemia treated with 5 daily doses of IL-1.alpha. (30 to 100 ng/kg) had no increases in peripheral blood counts or bone marrow cellularity (Walsh, C. E., et al., Br. J. Haematol 80:106 (1992)). IL-1 has been administered to patients undergoing various regiments of chemotherapy to reduce the nadir of neutropenia and thrombocytopenia.
Daily treatment with 40 ng/kg IL-1.alpha. from day 0 to day 13 of autologous bone marrow or stem cells resulted in an earlier recovery of neutropenia (median, 12 days; P&lt;0.001) (Weisdorf, D., et al., Blood 84:2044 (1994)). After 14 days of treatment, the bone marrow was significantly enriched with committed myeloid progenitor cells. Similar results were reported in patients with AML receiving 50 ng/kg/d of IL-1.beta. for 5 days starting at the time of transplantation with purged or nonpurged bone marrow (Nemunaitis, J., et al., Blood 83:3473 (1994)). Injecting humans with low doses of either IL-1.alpha. or IL-1.beta. confirms the impressive pyrogenic and hypotension-inducing properties of the molecules.
IL-1 Signaling Mechanisms
After binding to interleukin-1 (IL-1), the IL-1 receptor type I (IL-1RI) associates with the IL-1R Accessory Protein (IL-1RAcP) and initiates a signaling cascade that results in the activation of NF-kB, (Greenfeder, S. A., et al., J. Biol. Chem. 270:13757-65 (1995); Sims, J. E., et al., Science 241:585-9 (1988); Korherr, C.,et al., Eur. J. Immunol. 27:262-7 (1997); Wesche, H., et al., J. Biol. Chem. 272:7727-31 (1997); Freshney, N. W., et al., Cell 78:1039-49 (1994); and Martin, M., et al., Eur. J. Immunol. 24:1566 (1994)). Significant similarity exists between the IL-1R signaling pathway in mammals and the Toll signaling pathway in Drosophila. Toll, which shares sequence homology with the cytoplasmic domain of the IL-1RAcP, induces Dorsal activation (a homologue of NF-kB) via the adapter protein Tube and the protein kinase Pelle, (Galindo, R. L., et al., Development 121:2209-18 (1995); Norris, J. L. & Manley, J. L., Genes Devel. 10:862-72 (1996); Letsou, A., et al., EMBO 12:3449-3458 (1993); and Grosshans, J., et al., Nature 372:563-566 (1994)); significantly the recently identified IRAK (IL-1R Associated Kinase) is homologous to Pelle, (Cao, Z., et al., Science 271:1128-31(1996)). However, in mammalian cells, additional complexity is thought to exist based on the observation that multiple protein kinase activities coprecipitate with the IL-1RI (Singh, R., et al., J. Clin. Invest. 100:419 (1997); and Eriksson, A., et al., Cytokine 7:649 (1995)). Furthermore, given that in Drosophila the adapter protein Tube interacts with and regulates Pelle's activity, it is likely that analogous adapter/regulatory molecules might participate in IL-1 signaling. There is a need in the art to characterize molecules involved in the IL-1 signaling pathway.
Nuclear Factor Kappa B (NF-kB)
NF-kB is a member of a family of dimeric transcription factors made from monomers that have approximately 300 amino-acid Rel regions which bind to DNA, interact with each other, and bind the IkB inhibitors (for review, see Baeuerle and Baltimore, Cell 87:13-20 (1996)). Disregulation of NF-kB has been implicated in malignant transformation and hyperplasia (Gilmore et al., Oncogene 9:2391-2398 (1996)). NF-kB plays an important role in the antiviral response as a virus-inducible transcriptional regulator of .beta.-interferon, MHC class I, and inflammatory cytokine genes. NF-kB has also been shown to protect cells from pro-apoptotic stimuli (Beg et al., Nature 376:167-170 (1995)).